Construction Of CDNA Library Under Cold-stress And Clone And Functional Prediction Of Full-length FZF Gene In Chorispora Bungeana

Chorispora bungeana, a member of mustard family, is one of the typical subnival plants that can grow well in the freezing temperature so that it can be studied as the sample plant which is stress tolerance. After subjected to low temperature (-4℃) for 24h, the proline concentration and the soluble sugar concentration in Chorispora bungeana callus are both increased significantly. In contrast, the relative electrical conductivity (REC) and the malondialdehyde (MDA) concentration are not, which indicated that Chorispora bungeana can acclimatize the cold environment through regulate its own metabolism. cDNA library were constructed after exposure Chorispora bungeana callus to-4℃for 24h with the method of suppression subtractive hybridization (SSH) and cDNA array. There are 274 positive clones among 1536 monoclone in the cDNA library and 72 genes with the different function were screened. Of these 72 genes which are up-regulated or down-regulated under cold stress,25% (about 18) are transcription factor,15.3%(about 11) are metabolism-related or protein synthesis and degration-related,6.94%(about 5) are signal-related and photosynthesis-relaed, 4.17%(about 3) are stress related and 5.56%(about 4) are defense related. In addition, there are still 13.9%(about 10) are unkown proteins and 6.94%(about 5) are no match genes. These identified cDNAs encode proteins that may play a role in increasing cold resistant through Ca2+ signaling pathway, ABA-dependent signaling pathway and ABA-independent signaling pathway, which can maintain the cell membranes stable to keep the cell whole, increase the soluble substance to keep the normal osmotic pressure and enhance the photosynthesis to supply the energy. FZF full-length from Chorispora bungeana is acquired with rapid amplification of cDNA ends (RACE). The sequence of nucleotide and amino acid of FZF gene were highly homologic (86%identify and 91%similarity) in Chorispora bungeana and Arabidopsis thaliana. Further bioinformatics analysis revealed that 403 amino acid were encoded by 1212bp nucleotide with the molecular weight (MW) 45.7K.D and the isoelectric point (pI) 5.82. It's hydrophilic with two typical C2H2 zinc finger domain in N-side, but there's no transmembrane domain and signal peptide. It indicated that FZF from Chorispora bungeana works in the nuclear and regulates the downstream genes transcription as a transcription factor to enhance the cold resistance of Chorispora bungeana.